Abstract

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding.

Clinical, Histological, and Molecular Defects in Ablepharon-Macrostomia Syndrome and Barber-Say Syndrome(A) Face, shoulders, and hands of AMS-6.1, AMS-7.1, and AMS-7.2, demonstrating dysmorphic features detailed in . Shoulder photographs of AMS-6.1 and AMS-7.1 highlight Blaschko-like hyperpigmented banding indicative of mosaicism. Hands show mild cutaneous syndactyly and clinodactyly.(B) Electron microscopy of skin of unaffected control (1), AMS-6.1 (2), AMS-7.1 (3), and AMS-7.2. In (1), the elastin (asterisk) is ovoid in shape, whereas in (2), (3), and (4), the elastin appears elongated and, in some areas, fractured. Collagen fibers in (1) appear organized and are oriented in bundles. In (3) and (4), some collagen fibers appear in disarray (arrows) and show curved edges. In addition, some collagen fibers show variable diameters (6). Surrounding the elastin in (5) (AMS-7.1) and (6) (AMS-7.2) are flocculent and amorphous deposits that disrupt organization of collagen bundles. Scale bars represent 1,000 nm (1–4) and 500 nm (5 and 6).(C) Face, back, and hands of BSS-3.1 and BSS-4.2 showing dysmorphic features detailed in . Note hypertrichosis.(D) Electron microscopy of skin of BSS-3.1. Note the long and thin elastin fibers (asterisk) and collagen fibers in disarray (arrow) in (1). Similar to AMS, some collagen fibers show variable diameters (2). Surrounding the elastin in (3) are flocculent and amorphous deposits that disrupt organization of elastin and collagen bundles. Scale bars represent 1,000 nm (1) and 500 nm (2 and 3).

Mutations in the Basic Domain of TWIST2 Associated with AMS and BSS(A) Schematic of TWIST2 (GenBank: NP_476527.1) with locations of de novo missense variants identified in individuals with AMS and BSS.(B) Protein sequence alignment of vertebrate TWIST2 homologs. Residues in the basic domain affected by de novo variants are shaded gray.(C) Dimeric TWIST2 bHLH protein (gray) with bound DNA (yellow) model with inset. The p.Glu75 residue (red) is oriented toward the DNA major grove; this residue could be involved in hydrogen bonding with the first two nucleotides of the consensus E-box motif or positioning residue p.Arg78.

Effect of TWIST2 Mutations on HeLa Cell DNA Binding Sites and on Zebrafish Development and Gene Expression(A) Chromatin from HeLa cells overexpressing wild-type TWIST2 was subjected to ChIP-seq, identifying 630 DNA binding sites with a consensus sequence typical of an E-box motif.(B) ChIP-seq showed that the numbers of binding sites for p.Glu75Lys, p.Glu75Gln, p.Glu75Ala, and p.Gln77_Arg78dup TWIST2 were reduced compared to WT TWIST2 and that the mutants bound to many sites not shared with WT TWIST2.(C) Schematic of the zebrafish studies, involving embryos microinjected with mRNA at the 1-cell stage and either used at shield stage (6 hpf) for RNA-seq or left to grow until approximately 30 hpf for phenotypic characterization.(D) Appearance of mild, moderate, and severely affected embryos, and quantification of the phenotypes induced at 30 hpf by overexpression of WT, p.Glu75Lys, and p.Glu75Gln hTWIST2. Embryos were injected at the 1-cell stage with 10 pg mRNA. hTWIST2 variants induced defects in head structures and failure of the posterior end of the embryo to extend properly. The p.Glu75Lys and p.Glu75Gln mutants induced stronger developmental defects than wild-type hTWIST2 mRNA.(E) GO term analysis on ranked gene lists from RNA-seq for p.Glu75Lys and p.Glu75Gln mRNA. Extracellular matrix, membrane, and cytoskeleton proteins are downregulated.